The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane

Abstract

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins. We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.

Figure 1

Diagram showing the correlation of the genetic and physical maps in the unc-112 region of LG V. The top region of the figure is a partial genetic map showing the relative positions of the three genes and two deficiencies used in this study. The location of sequenced cosmids in this region of the genome relative to the two deficiency breakpoints is shown below. Asterisks indicate the approximate regions of the various cosmids that were tested for amplification by PCR. The unc-112 gene is located on cosmid C47E8.

Figure 3

Comparison of the predicted amino acid sequences of UNC-112, the human Mig-2 protein and the CG7729 and CG14991 proteins in D. melanogaster. The nematode, fly, and human proteins are ∼60% similar (∼41% identical) over their entire length, and share a short region of homology with talin and other members of the FERM superfamily (aa 288–488 of UNC-112). The amino acid sequences were aligned using the Clustal W program. Identical amino acids are shaded, and similar amino acids are boxed. The functional UNC-112::GFP protein has the GFP inserted between Arg28 and Ser29 of the UNC-112 amino acid sequence.

Figure 2

Diagram of a 7.5-kb genomic DNA fragment containing the C47E8.7/unc-112 ORF. The six exons are represented as boxes. The intron/exon boundaries shown here have been confirmed by sequencing the full-length YK12c6 cDNA clone (data are available from GenBank/EMBL/DDBJ under accession number 217185). The sequence alterations corresponding to four unc-112 mutations are also indicated. Four exons encoding the GFP protein were introduced into the first intron of the unc-112 ORF to obtain the pDM#211 construct.

Figure 4

Comparison of the homologous amino acid sequences of UNC-112 and members of the FERM protein superfamily. Alignment of aa 288–488 of UNC-112 with (A) aa 173–369 of the human talin sequence, and (B) aa 107–293 of the mouse band 4.1 sequence and aa 58–258 of the chicken ezrin sequence. The UNC-112 sequence is more similar to talin (∼53% homology) than to band 4.1 and ezrin (∼33% homology). Note the very high homology between the first 52 amino acids of the alignment of UNC-112 and talin (50% identity; 65% similarity). The sequences were aligned using the Clustal W program. Identical amino acids are shaded, and similar amino acids are boxed.

Figure 5

Localization of UNC-112::GFP fluorescence and MH25 (PAT-3/β-integrin) immunofluorescence in adults and embryos. (A) UNC-112::GFP fluorescence in adult body wall muscle of a rescued unc-112(st581); raEx16[unc-112::GFP; rol-6(su1006)] hermaphrodite. (B-D) colocalization of UNC-112::GFP (green) with PAT-3/integrin (red) in the same body wall muscle cell of a +/+; raEx16[unc-112::GFP; rol-6(su1006)] hermaphrodite; (B) UNC-112::GFP fluorescence, (C) MH25 immunofluorescence, (D) both GFP and MH25. Arrowheads indicate the M-line; small arrows indicate dense bodies; and large arrows indicate adhesion plaques between muscle cells. (E-G) colocalization of UNC-112::GFP (green) with PAT-3/integrin (red) in the body wall muscle of a 1.5-fold +/+; raEx16[unc-112::GFP; rol-6(su1006)] embryo. (E) UNC-112::GFP fluorescence, (F) MH25 immunofluorescence, (G) both GFP and MH25. All images are projected to show a single muscle quadrant. Bar, 10 μm.

Figure 6

Perlecan, integrin, and vinculin localization in wild-type and unc-112(st581) embryos. Wild-type (N2) embryos (A, C, E, and G) and unc-112(st581) embryos (B, D, F, and H) stained with the MH2 mAb which recognizes UNC-52/perlecan, the MH25 mAb which recognizes PAT-3/β-integrin, and the MH24 mAB which recognizes DEB-1/vinculin. A and B show staining with MH2; C, D, E, and F show staining with MH25; and G and H show staining with MH24. A′–H′ are magnifications of A–H, respectively. The wild-type embryos in A, E, and G are at the threefold stage of embryonic development. The mutant embryos in B, F, and H have arrested at the twofold stage of embryogenesis, but are comparable in age to the threefold wild-type embryos. The two embryos in C and D were stained at an earlier stage (∼350 min after the first cell division), before the mutant embryo has arrested development. The unc-112(st581) mutant embryo can be identified at this early stage by a gap in the ventral quadrant which is detectable by ∼300 min after the first cell division. All of the mutant embryos were obtained as segregants from unc-112(st581); raEx16[unc-112::GFP; rol-6(su1006)] hermaphrodites. All of the panels show lateral views of the embryos except C and D which show dorsal views. Note the disorganized staining in the mutant embryos in F and H. In all cases, images have been projected from a full Z-series to show two muscle quadrants. The arrows in A–H indicate the regions of each embryo that have been magnified in A′–H′. The arrows in E′–H′ indicate adhesion structures. Bar, 10 μm.

Figure 7

UNC-112 localization in mutant embryos. DM5.6 immunofluorescence and UNC-112::GFP fluorescence in wild-type (A and B), unc-44(e362)deb-1(st555) IV (C and D), unc-52(ra401) II (E and F) and pat-3(st564) III (G and H) embryos. A, C, E, and G show staining with the DM5.6 mAb. B, D, F, and H show GFP fluorescence. B′, D′, F′, and H′ are magnifications of B, D, F, and H, respectively. All of the embryos are at the ∼1.5-fold stage of embryonic development. The mutant embryos were obtained as segregants from heterozygous parents carrying the raEx16[unc-112::GFP; rol-6(su1006)] transgenic array. The genotype of the wild embryo in A and B is either unc-52(ra401)/+; raEx16 or +/+; raEx16. All panels show lateral views of the embryos. The images have been projected from a full Z-series to show one dorsal and one ventral muscle quadrant. The arrows in B, B′, D, D′ F, F′ G, and G′ indicate UNC-112::GFP fluorescence. Note the absence of any organized myosin staining in C, E and G, or GFP fluorescence in F, F′, H, and H′. Bar, 10 μm.